Project 1 aims to induce tolerance of porcine kidneys in baboons by co-transplantation (Tx) of vascularized thymus. We have induced donor-specific T cell unresponsiveness and prevented anti-donor elicited antibodies (Abs), but have not achieved long-term survival of xenograft kidneys. During the previous project period, we identified two major obstacles to this goal and developed strategies to overcome them. These are: (i) early loss of xeno- thymokidneys (TKs) due to activation of latent porcine CMV (pCMV); long-term graft survival was restored by elimination of pCMV through cesarean section of donors; and (ii) development of severe proteinuria; this problem could be delayed (but not prevented) by preventing SMPDL-3b-dependent disruption of pig podocytes through treatment with Rituximab in the peri-Tx period. The remaining major obstacles addressed in the current proposal are: 1) eventual development of nephrotic syndrome due to progressive proteinuria; and 2) development of infections due both to loss of immunoglobulins from proteinuria and presumably insufficient recovery of protective T cell immunity from porcine thymic grafts. Based upon the data developed by this team (Projects 2, 3, and 4), we have designed innovative strategies to overcome these obstacles. In Aim 1, we will first identify the mechanism responsible for continuing proteinuria despite Rituximab treatment and develop effective treatment strategies. We have found both loss of SMPDL-3b and upregulation of porcine CD80 on podocytes to be associated with xenograft nephropathy. Our preliminary data indicate that Rituximab, which binds to porcine podocyte SMPDL-3B and Belatacept, which inhibits CD80 activation, can both partially stabilize protein loss. The latest transplant with this combined therapy currently survives >70 days, with normal creatinine levels and only minimal proteinuria. Our data suggest that CD47 and SIRP-alpha incompatibility between species may promote innate immune activation that culminates in podocyte activation. In Aim 1, we will overcome proteinuria by optimizing Rituximab/CTLA-4 Ig therapy and using hCD47 transgenic (Tg) GalT-KO pig TK donors to prevent baboon macrophage activation. In Aim 2, we will achieve xenograft tolerance with more rapid reconstitution of host- and donor-restricted protective T cell immunity. Studies in Project 3 demonstrated limitations in human T cell function and homeostasis following development in a pig thymus. We will address the hypothesis that these abnormalities will be corrected by adding recipient TEC to the porcine thymus graft combined with mixed chimerism. We will now optimize the construction of hybrid thymic grafts, assess their impact on immune function and combine this protocol with strategies developed in Project 2 to achieve durable mixed xenogeneic chimerism (Aim 3). The combination of hybrid thymic grafting and durable mixed chimerism will assure tolerance of both innate and adaptive immune responses. The combined approach will also optimize functions of T cells providing protective immunity against infections for the donor graft and recipient and of Tregs that protect against residual donor-reactive T cells and autoimmunity, respectively.